Revolutionary advances in molecular imaging technologies have allowed researchers to carry out quantitative examination of molecular dynamics and cell signaling in living cells (Non Patent Literature 1). As described in Non Patent literature 2, one of the imaging technologies with lighting protein is circular permutation (CP) of fluorescent proteins such as green fluorescent protein (GFP) for construction of probes. CP of GFP is a mutation in which the polypeptide of GFP is dissected and the N- and C-terminal fragments are inversely linked.
GFP has a conformation whose shape is a monolithic cylindrical symmetry wherein hydrophobic amino acids are serially arranged in a lattice manner. The principle of the circularly permutated probe with GFP is as follows. First, the fluorescence intensity from GFP is suppressed by water molecules accessing to the internal chromophore via a partially cleaved hole of GFP. The ligand recognition protein cofused to GFP closes the cleaved hole in response to a specific ligand. This causes the water molecule to be expelled from the chromophore, which results in enhancement of the fluorescence intensity. In this manner, the variation in the fluorescence intensities of GFP in the cells visualizes dynamics of molecular events in the cells.
The convention circular permutation of the fluorescent proteins was valid (i) only when the fluorescent proteins tolerates to insertion of a variety of proteins and (ii) only when the original N- and C-termini are spatially close enough to be linked (Non Patent Literature 13). The circular permutation of GFP required great skills because of their monolithic cylindrical symmetry and complexity of their protein strands. Thus, it was generally difficult to perform circular permutation.
Conventionally, the fluorescent proteins are suffered from an intrinsic problem that autofluorescence-caused elevation of background intensity. Further, fluorescent proteins requires an external light source and a relatively large instrumentation such as a fluorescence microscope for signalizing fluorescence. Autofluorescence inevitably causes an elevation of background intensity and poor signal-to-noise contrast in case of Yellow Camelleons (Non Patent Literatures 2 through 4) for example. In addition, the obtained results from fluorescent proteins are qualitative rather than quantitative because of the limited number of analyzable cell population at once (Non Patent Literature 5).
As a complement for the fluorescence proteins, bioluminescent proteins have been utilized in designing a new molecular probing system (Non Patent Literatures 5 through 9): e.g., providing a whole cell investigation; low background intensity; no external light sources.
Further, the present inventors demonstrated a single-molecule-format bioluminescent probe for imaging androgenic activities of ligands (Non Patent Literatures 10 and 11). The fundamental concept of single-molecule-format bioluminescent probe is to design a single-chain protein, in which all the components for signal recognition and light emission are integrated. In the single-molecule-format bioluminescent probe described in Non Patent Literatures 10 and 11, (i) a target ligand recognition protein and (ii) its interacting protein are sandwiched between the N- and C-terminal fragments of a dissected luciferase. The target ligand recognition protein activated by a ligand triggers an intramolecular complementation between the adjacent N- and C-terminal fragments in the bioluminescent probe. This complementation resulted in recovery or termination of the activities (exhibiting bioluminescence) of luciferase. The luminescence intensities were taken as a measure for visualizing molecular dynamics of ligand recognition proteins in cells.